Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.     

Bull influences on conception percentage and calving date in Angus Hereford, Brahman and Senepol single-sire herds

Bull breeding soundness parameters, semen characteristics and sexual behavior were evaluated for effects on reproductive performance in single-sire beef herds. A total of 155 cow herds (Angus, 50 herds; Hereford, 40 herds; Brahman, 46 herds; and Senepol, 19 herds) bred to bulls of the same breed were observed for 8 yr. All bulls produced adequate quality semen and had scrotal circumference (SC) >/= 30 cm. Reproductive performance was evaluated by the conception rate (CON), conception rate during the first 21 d of the breeding season (21dCON), mean calving date (MCD), and mean calving date of the first half of the herd to calve (HHCD). Correlations were determined between breeding soundness parameters and reproductive performance for all bulls combined, by breed, and by age. The Cp statistic was used to select models for the effects of parameters on CON, 21dCON, MCD and HHCD. Breeding season length and breed had significant effects. The percentages of normal cells, proximal droplets, detached heads and the semen score (motility plus percentage of normal cells) had a significant effect on CON when all bulls were considered. After the effect of season was deleted, the most significant parameter affecting CON in the Brahman was the percentage of detached sperm heads. In the Angus, motility was significantly correlated with all reproductive performance indices. In the Hereford, breeding soundness examination score (BSE) was positively correlated with 21dCON.     

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Hormonal stimulation of tyrosinase activity in human foreskin organ cultures

Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining. Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle⁴, D-phe⁷]-alpha MSH (10⁻⁸ M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E₁ produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D₃ (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation in human skin. This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST) and by a research grant from the Presbyterian Health Foundation.     

Toward Practical Biological Control of Parasitic Nematodes in Domestic Animals

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.